Advances in Applied Biotechnology Part 10 ppt

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degradation of α-amylase-A522-PreS2 by the inactivation of IspA in the KA8AX strain.
However, the productivity of α-amylase-A522-PreS2 in the ten-protease deficient mutant
was almost same as that in the KA8AX strain. A search in the GenoList database for B.
subtilis 168 genome ( of proteases
and peptidases revealed the presence of 31 known and 11 putative proteases, and 38 known
and 12 putative peptidases, respectively. Section 2.2 describes the investigation of
membrane-bound proteases involved in protein degradation.

Fig. 5. Western blot analysis of the α-amylase-A522-PreS2 hybrid protein in the extracellular
fractions of Dpr7, Dpr8, and KA8AX. (A) Western blot analysis was carried out to detect α-
amylase-A522-PreS2 with the anti-PreS2 antibody. Culture supernatants from Dpr7 (lanes 1-3),
Dpr8 (lanes 4-6), and KA8AX (lanes 7-9) were collected after 25, 50 h, and 75 h of cultivation,
and subjected to Tricine-SDS-PAGE and Western blotting, as described in the Materials and
Methods. Proteins from the culture supernatants (equivalent to 1 µl) were applied to each lane.
The arrowhead indicates the position of α-amylase-A522-PreS2. The times of harvest of
supernatants are shown at the top. (B) The relative α-amylase-A522-PreS2 protein amounts
were compared on the basis of band intensities on Western blots (the amount of α-amylase-
A522-PreS2 at 50 h in the Dpr8 strain was set to 100%). The presented results are the average of
three individual experiments. Error bars correspond to the standard errors of the means
(SEM). Lane numbers in panel A correspond to those in panel B.

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Fig. 6. The α-amylase-A522-PreS2 hybrid protein was degraded by AprX. (A) Zymography

of supernatants from the KA8AX(pDG-AprX) strain. Lane 1, without IPTG; lane 2, with
IPTG. (B) Western blot analysis of degradation of α-amylase-A522-PreS2 by AprX. AprX
from KA8AX (pDG-AprX) mutant cells, cultured for 4 h with or without 1 mM IPTG was
prepared as described in the Material and Methods. α-Amylase-A522-PreS2 from 10 µl
supernatants of the KA8AX (pTUBE522-preS2) mutant (at 75 h cultivation) was mixed with
10 µl of AprX solution. After incubation at 37ºC for 60 min, PMSF (final concentration, 10
mM) was added to the samples to stop the reaction. Western blot analysis was carried out to
detect α-amylase-A522-PreS2 with the anti-PreS2 antibody; +, addition of 1 mM IPTG
(AprX); -, no addition. The reaction mixture (equivalent to 1 µl) was applied to each lane.
The arrowhead indicates the position of α-amylase-A522-PreS2. (C) The relative amounts of
α-amylase-A522-PreS2 were obtained by comparing the band intensities on Western blots
(the α-amylase-A522-PreS2 amount in lane 1 was set as 100%). Lanes 1 to 6 in panel C
correspond to lanes 1 to 6 in panel B.
2.2 The effect of HtrA and HtrB on the degradation of secreted proteins
In this section we describe the effects of membrane-bound proteases and a two-component
system on degradation of secreted proteins, and transcriptional regulation of the membrane-
bound protease genes.
2.2.1 Cell envelope-associated quality control proteases
In B. subtilis, the accumulation of misfolded proteins at the membrane-cell wall interface is
sensed by the CssR–CssS two-component system, which consists of the membrane-
embedded sensor kinase, CssS and the response regulator, CssR (Hyyryläinen et al., 2001).
This system responds to general protein secretion stresses, which can be triggered by either
homologous (e.g., overproduction of LipA) or heterologous (e.g., overproduction of AmyQ
and hIL-3) proteins, and consequently activates the transcription of the monocistronic htrA
and htrB genes (Darmon et al., 2002; H. Westers et al., 2006; Hyyryläinen et al., 2007). HtrA
and HtrB are membrane-bound serine proteases that are responsible for the degradation of
misfolded proteins, and can thereby rescue the cell from a lethal accumulation of misfolded
proteins in the cell envelope. In addition, HtrA has a dual localization, because it can be
detected in the membrane-associated cellular fraction as well as the growth medium.
Therefore, HtrA has a chaperone-like activity that might assist misfolded proteins in

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recovering their conformation, while also targeting unsuccessful protein for degradation
(Antelmann et al., 2003). Induction of htrA and htrB expressions is responsive to secretion
stress in a manner dependent on the CssRS two-component system. In addition, htrA and
htrB expressions are negatively autoregulated and reciprocally cross-regulated (Noone et al.,
2000, Noone et al., 2001). Therefore, the absence of HtrA leads to the increased synthesis of
HtrB, and vice versa (Noone et al., 2001).
2.2.2 High-level lipase A (LipA) production in eleven proteases mutant
We examined the production of lipase A (LipA) of B. subtilis (van Pouderoyen et al., 2001), as a
valuable model for industrial enzyme production, in a nine-protease-deficient B. subtilis strain.
Therefore, we constructed the pHLApm plasmid, in which LipA with the promoter and
ribosomal binding site of an alkaline cellulase gene, egl-237 (Hakamada et al., 2000) was cloned
into pHY300PLK (Takara, Japan). LipA was overproduced in B. subtilis. Cells carrying
pHLApm were cultured in modified 2xL broth for 12, 24, 36, 48, 60, and 75 h. The productivity
of LipA in the supernatants from cultures of the 168 and Dpr9 (in which nine genes encoding
eight extracellular proteases and AprX were precisely and completely deleted from the
chromosome) strains was calculated based on the activity of LipA (Fig. 7). In 24 h cultivation,
the production level of the LipA in 168 and Dpr9 could be obtained at 860 mg/L, an excellent
yield which is 1.4-times higher than that of previously reported (Lesuisse et al., 1993). After 24
h, the amount of LipA markedly decreased in the 168 strain (Fig. 7). In contrast, degradation of
LipA in the Dpr9 was effectively inhibited, compared with the 168 strain. However, after 36 h,
the production of LipA in Dpr9 was reduced by approximately 10% (Fig. 7). These results
showed that LipA was also degraded in the Dpr9 strain. Overproduction of both homologous
(LipA) and heterologous (AmyQ and hIL-3) proteins induces the expression of htrA and htrB
by the CssRS system (Darmon et al., 2002; H. Westers et al., 2006). From the currently available
data, it seems most likely that limitation of both proteases of HtrA and HtrB improved the
yield of heterologous proteins (Vitikainen, M., H. L. et al., 2005). To confirm the effect of HtrA

and HtrB on the degradation of secreted proteins, we examined the production of LipA of B.
subtilis in the htrA and/or htrB deficient B. subtilis strains. We constructed Dpr9∆htrA,

0
20
40
60
80
100
120
140
0 20406080
Time (h)
Relative activity (%)

Fig. 7. Time course of LipA activity in the Dpr9 mutant. Cells were cultured in modified 2xL
broth at 30ºC. The accumulation of LipA in the culture medium was measured at various
incubation times. Open circles, wild type strain; closed triangles, Dpr9 mutant.

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Dpr9∆htrB, and Dpr9∆htrA/B (with eleven inactivated proteases), and evaluated each strain
for the production of LipA. No effect on LipA production was observed in Dpr9∆htrA and
Dpr9∆htrB. However, the production of LipA by the Dpr9∆htrA/B strain was at 1100 mg/L,
which is 1.2-times higher than that of the Dpr9 strain (Fig. 8). These results suggest that
inactivation of both htrA and htrB, as well as the nine proteases, has improved the
productivity of B. subtilis for the production of LipA.

Fig. 8. Enhanced productivity of LipA in the absence of both htrA and htrB. Cells were
cultured in modified 2xL broth at 30ºC. The accumulation of LipA in the culture medium
was measured at 48 h. The relative activities of LipA are shown (the amount of Dpr9 was set
to 100%).
2.2.3 Transcriptional regulation of htrB and htrA by reciprocal cross regulation
We predicted that there was no difference between the productivities of LipA in the
Dpr9∆htrA and Dpr9∆htrB strains, because the inactivation of either htrA or htrB results in a
compensating overexpression of the other gene (Noone et al., 2001). To confirm that the
overexpressions of htrA and htrB are caused by the inactivation of the other gene, we
examined the level of expression of the htrB-lacZ fusion for the Dpr9∆htrA mutant, as well
as the similar expression of the htrA-lacZ fusion for the Dpr9∆htrB mutant. Cells carrying
pHY300PLK (control) and pHLApm (LipA overexpression) were cultured in modified 2xL
broth for 48 h. As shown in Table 1, Dpr9∆htrA cells harboring pHLApm transcribed htrB-
lacZ at a 4-fold increased level, compared with Dpr9 harbouring pHLApm (from 0.51 to 2.30
U). Similarly, a 10-fold increase in the htrA-lacZ expression level was observed in the
Dpr9∆htrB mutant (from 0.41 to 4.26 U). The expressions of htrB-lacZ and htrA-lacZ also
demonstrated reciprocal cross regulation in cells carrying pHY300PLK. These observations
suggest that the overexpression of htrB in Dpr9∆htrA and of htrA in Dpr9∆htrB might affect
LipA production. The expression level of htrB-lacZ in LipA-producing Dpr9 was 2.4-times
higher than that of non-LipA-producing Dpr9 (Table 1). There was almost no change in the
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expression level of htrA-lacZ, between Dpr9 cells harboring pHLApm and Dpr9 cells
harbouring pHY300PLK. The expression of the htrB-lacZ reporter gene fusion has previously
been shown to be more sensitive to secretion stress than the htrA-lacZ reporter gene fusion
(Hyyryläinen et al., 2001). These results suggest that Dpr9 produced LipA in weak response
to secretion stress.
Expressed gene Strain Plasmid Expression

a

htrB-lacZ Dpr9 pHY300PLK 0.22±0.01
Dpr9∆htrA pHY300PLK 1.46± 0.11
Dpr9 pHYLApm 0.51± 0.11
Dpr9∆htrA pHYLApm 2.30± 0.02
htrA-lacZ Dpr9 pHY300PLK 0.38± 0.03
Dpr9∆htrB pHY300PLK 1.29± 0.01
Dpr9 pHYLApm 0.41± 0.03
Dpr9∆htrB pHYLApm 4.26± 0.02
a
One activity unit is defined as 1 nmol of O-nitrophenyl-ß-D-galactopyranoside hydrolysed per min per
µg of OD
600
. The results presented are the average of three individual experiments. Plus/minus values
represent standard deviations.
Table 1. Expression of transcriptional fusions between the htrA and htrB promoters and lacZ
reporter gene in various genetic backgrounds.
3. Conclusion
This chapter focused on biotechnological approaches to optimization of heterologous
protein and enzyme production by multiple protease-deficient mutations in B. subtilis.
Section 2.2 described the identification of AprX protease using gelatin zymography and the
effects of AprX on heterologous protein production. The nine-protease-deficient KA8AX
strain (lacking nine genes encoding eight extracellular proteases and AprX) effectively
prevented proteolysis of α-amylase-A522-PreS2 [PreS2 antigen of human hepatitis B virus
(HBV) fused with the N-terminal 522 amino acids of B. subtilis α-amylase] in the late
stationary growth phase and improved the yield of the fusion protein. In addition, AprX
was detected in the culture medium due to leakage on cell lysis during the late stationary
growth phase. Section 2.3 described that the inactivation of nine-proteases and both htrA
and htrB (resulting the Dpr9∆htrA/B mutant) improved the productivity of LipA in B.

subtilis. In particular, the productivity of the LipA in the Dpr9∆htrA/B strain was 1100
mg/L, an optimal yield which is 1.8-times higher than that of previously reported. There
was no difference in the productivities of LipA in the Dpr9∆htrA and Dpr9∆htrB strains,
compared with that of Dpr9. Because the transcriptions of htrA and htrB are controlled by
reciprocal cross regulation, overexpression of htrB in the Dpr9∆htrA strain and of htrA in the
Dpr9∆htrB strain might affect LipA production. The previous approach for effective protein
production was to generate a strain which has the inactivation of eight extracellular
proteases in B. subtilis as the host. We reported that AprX leaked outside of cells, and
HtrA/HtrB membrane-bond proteases of B. subtilis were also key proteases involved in the
degradation of natural and heterologous proteins. In addition, nine- or eleven-protease-
deficient strains of B. subtilis were helpful in improving protein productivity. Our findings,
described in this chapter should contribute to the generation of hosts to be further optimized
for protein production.

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4. Acknowledgment
We would like to thank Mr. Keiji Endo, Mr. Kazuhisa Sawada, Dr. Koji Nakamura, Dr.
Yasutaro Fujita, Dr. Fujio Kawamura, and Dr. Naotake Ogasawara for useful advice and
discussions, and Dr. Hiroshi Kakeshita and Dr. Kunio Yamane for their generous gift of
plasmid of pTUBE522-PreS2, and for useful advice and discussions. This work was
supported by the New Energy and Industrial Technology Development Organization
(NEDO).
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9
The Development of Cell-Free
Protein Expression Systems and
Their Application in the Research on
Antibiotics Targeting Ribosome
Witold Szaflarski, Michał Nowicki and Maciej Zabel
Department of Histology and Embryology, Poznań University of Medical Sciences,
Poland

1. Introduction
There is a little doubt that increasing developments of protein synthesis are in high demand.
Not only proteins are participants in all biochemical processes of the living cell, continually
accelerating advances in proteomics, (i.e. the science of proteins and their reciprocal
interactions in the cell) are increasingly underscoring the need to perfect techniques that
facilitate the production of specified proteins at an industrial scale that meets the necessary
standards of purification (Kim and Kim 2009). Investigations that have built the foundation
for such protein production have largely originated from discoveries in the middle of the
last century. Such advances firstly elucidated new cellular environments of protein
production. Subsequent developments focused on the specificity of protein synthesis and
the general efficiency of production has been developed largely by genomic analysis and
genetic recombination.
Several in vitro systems of protein synthesis are commercially available worldwide. Many of
these methods are categorized according to the derivation of their extracts, from either
prokaryotic cells such as Escherichia coli (E. coli) or, alternatively, eukaryotic cells such as
wheat germ or rabbit reticulocytes. While such extracts can be enriched by cofactors that
enhance the efficacy of protein biosynthesis, there are obvious limitations to such systems.
An important criterion involves also simplicity of the system and its potential application:
(1) simple systems, such as synthesis of phenylalanine homopolymer (poly(U)-dependent
poly(Phe) expression) are generally applied in studies that analyze protein biosynthesis itself
and on factors which block the process, i.e. antibiotics. This is in contrast to (2) the complex
systems that are able to link transcription and translation into a single system.
The most advanced cell-free system based on the application of semi-permeable membrane
allowing the concentration of reaction compartment during the work with ribosomes. Such
membrane separates the feeding compartment where energy-rich molecules are deposed
and can be moved to the reaction compartment with a simple diffusion. Moreover, such a
feeding compartment is a suitable space where by-products potentially interfering with the
biosynthesis can be deposed.

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Recently, many of different cell-free based systems are available and the customer can select
the most suitable for the specific application. Here, we described the most popular systems
and we demonstrated how these systems can be utilized to study interactions between
antibiotics and the ribosome.
1.1 The beginning of cell-free protein synthesis
In 1950s, several research teams independently demonstrated that protein biosynthesis can
take place even after disintegration of the cell membrane (Siekievitz and Zamecnik 1951;
Borsook et al. 1950; Winnick 1950; Gale and Folkes 1954). Thus, the isolated cytoplasm has
been found to comprise the entire set of components necessary to conduct protein
biosynthesis. As first, Zamecnik prepared fully active cell-free system based on
mitochondrium-isolated ribosomes from an animal (Littlefield et al. 1955; Keller and
Littlefield 1957). The team further demonstrated that the reactions were dependent on the
supply of high energy molecules, such as ATP and GTP. The first in vitro systems of protein
synthesis based on isolated bacterial ribosomes were designed independently by two teams,
German (Schachtschabel and Zillig 1959) and American (Lamborg and Zamecnik 1960).
However, both of them were only capable of translating endogenous mRNAs, what was
their main limitation. Nevertheless, this discovery provided a proof that extracellular
biosynthesis was possible at all and consequently it provided a new approach to synthesize
proteins and to study molecular mechanisms of protein biosynthesis. An open nature of the
in vitro systems was very attractive especially to the latter approach.
The discovery of protein expression systems on the template of exogenous mRNA
molecules significantly extended applications of extracellular protein biosynthesis. The
achievement took place in 1961 in the laboratory of Nirenberg and Matthaei (Nirenberg
and Matthaei 1961). A short incubation at the physiological temperature of around 37ºC
proved sufficient to remove endogenous mRNA molecules from ribosomes. Free
ribosomes obtained from the procedure were subsequently used for protein synthesis on
the template of exogenous mRNA molecules. Of great importance, the ribosomes could be
"programmed" by synthetic mRNA molecules. The technique of Nirenberg became the

classical system of extracellular protein synthesis and, taking advantage of it, its
originator deciphered the genetic code, for which he received the Nobel prize in 1968. In
the subsequent systems, additional procedures of purifying ribosomes from endogenous
mRNA molecules were applied to DEAE cellulose, permitting the separation ribosomes
from free nucleic acids via chromatography.
Incubation of ribosomes, preceding the proper protein biosynthesis and conducted in the
same manner as in the technique of Nirenberg, was later successfully applied in
eukaryotic in vitro systems. Extracts of animal cells enriched with purified ribosomes
conducted efficient protein biosynthesis. The technique was again successful using the
template of exogenous mRNA molecules (Schreier and Staehelin 1973). During
approximately the same timeframe, investigators applied this capacity to extracts of
wheat germs and, of great interest, found that the endogenous as opposed to exogenous
expression of mRNA molecules manifested naturally low levels of protein (Marcus, Efron,
and Weeks 1974; Roberts and Paterson 1973; Anderson, Straus, and Dudock 1983). Other
techniques of eliminating endogenous mRNA were based on application of calcium ion-
dependent bacterial RNAse, used to augment the efficiency of protein expression system
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in lysates of erythrocytes (Jackson and Hunt 1983; Merrick 1983; Pelham and Jackson
1976) as well as in other lysates originating from animal cells (Henshaw and Panniers
1983). In the 1980s, it was subsequently found that bypassing the expression of
endogenous mRNA molecules significantly improved the efficiency of extracellular
protein expression systems.
1.2 Simple systems based on synthesis of protein homopolymers
In such systems, the principal homopolymeric system involves synthesis of polyalanine on
the template of poly(U) chain (poly(U)-dependent poly(Phe) synthesis). A buffered medium
containing free ribosomes and the remaining components necessary for a translation
reaction with the template of poly(U), the polyuridine homopolymer is added. The poly(U)

template has the capacity to bind a ribosome without involvement of the Shine-Dalgarno
sequence (sequence on mRNA which binds to the region of 16S rRNA) also has the ability to
"program" the ribosome for synthesis of poly(Phe). Efficiency of the optimised systems of
polyphenyloalanine synthesis may reach 300 amino acid incorporations per ribosome,
which represents a significant achievement allowing for a unitemporal and complete
analysis of all protein biosynthesis components (Szaflarski et al. 2008). In contrast, the first
attempts of the type, performed in 1950s and 1960s resulted in merely 2-5 amino acid
incorporations per ribosome.
The homopolymeric system was prepared by isolation of two cellular fractions, which were
subsequently enriched in high-energy molecules, free amino acides and poly(U)-mRNA,
providing the template. The fractions were obtained from bacterial extracts, which were
fractionated by centrifugation (for a detailed description of ribosome isolation see (Blaha et
al. 2000)). The so-called fraction S30 (obtained by centrifugation at approximately 30,000
rpm for 24 h) was rich in ribosomes and was used to purify free ribosome subunits in a
sucrose gradient (centrifugation at around 45,000-60,000 rpm for 15 h). Ribosomes prepared
in this manner were incubated at the temperature of 37ºC in a buffer containing, for
instance, Mg
2+
ions at the concentration of 4.5 mM in order to obtain complete correct 70S
ribosome structure capable of performing protein synthesis.
S100 fraction was obtained from supernatant of the S30 fraction and it provides the source of
protein factors indispensable to conduct translation (i.a., initiation factors: IF1, IF2, IF3,
specific aminoacyl-tRNA synthetases, elongation factors: EF-Tu, EF-G, EF-Tu).
The reaction of polyphenylanaline synthesis represents a simple and widely used technique
in several varieties. Instead of a poly(U) template, a poly(A) template can be used, enabling
the synthesis of polylysine. Unfortunately, however, the polymer was poorly soluble in
water; this property markedly restricts applicability of the system at a broader scale.
Nevertheless, application of certain detergents permits its application in studies as seen in
previous experiments conducted with the functional analysis of two antibiotics (pactamycin
and edein), representing inhibitors of protein synthesis (Dinos et al. 2004). Here, the

incorporation of near-cognate lysine instead of phenylalanine on the template of poly(U) can
be precisely measured using double radioisotope labeling. If any antibiotic impacts on the
translation accuracy (for example aminoglycoside paromomycin) it can be confirmed by
detection of higher incorporation of lysine. Followed that technique edein was found to be

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an error-prone antibiotic in contrast to pactamycin which did not induce any miscoding
(Dinos et al. 2004).
1.3 Biosynthesis of protein in a couple transcription-translation system
Nirenberg and Matthaei (Matthaei and Nirenberg 1961) again were the first to describe the
DNA dependence of the bacterial extracellular synthesis of protein. The dependence was
corroborated by synthesis of a protein on the template of endogenous DNA molecules.
Another group of investigators extended synthesis of endogenous proteins by application of
exogenous DNA, which originated from a bacteriophage (Byrne et al. 1964; Wood and Berg
1962). Unfortunately, the systems manifested a relatively poor efficiency using either
endogenous or viral DNA. Moreover, they were accompanied by a non-specific expression
of cellular and bacteriophage proteins. However, the continuing improvements of the joint
transcription and translation system resulted in its dissemination; with it ultimately
becoming a significant laboratory tool (Lederman and Zubay 1967; DeVries and Zubay
1967).
In the improved system, suggested by Zubay, a preliminary bacterial extract was subjected
to incubation in order to degrade mRNA and DNA molecules by cellular nucleases (Zubay
1973). The system gained popularity due to the ease of its preparation, stability of
components and a relatively high efficiency. In the system designed by Gold and Schweiger
ribosomes were isolated from cellular extracts to their homogenous form and so prepared
ribosomes were supplemented with a cytoplasmic fraction, cleared of nucleic acids by ion-
exchange chromatography (Schweiger and Gold 1969, 1969, 1970). Such a preparation of
components for extracellular protein synthesis produced a remarkable reduction in the non-

specific expression of protein. The troublesome procedure, however, remained the
disadvantage of the system.
2. Contemporary systems of the cell-free protein expression
The 1980s and 1990s witnessed development of the in vitro systems in the form of
optimization of cellular extracts, including the application of bacterial strains lacking the
genes that code enzymes of endonuclease type (RNases) (Zaniewski, Petkaites, and
Deutscher 1984). Such reaction mixes allowed researchers to keep bacteria in the reactive
mix for a much longer period of time. Contemporary systems are characterized by a high
mRNA level even after 24 h of the reaction (Iskakova et al. 2006). In 1990s the developing
methods of genetic engineering and bioinformatics produced tremendous advances inside
in vitro systems. One tremendous achievement was the ability to obtain data on structure of
mRNA transcripts and on the effect of the structure on the efficiency of biosynthesis. The
spatial structure of mRNA and primarily the sequence located at the 5’ terminus, proved to
be very important (Graentzdoerfer et al. 2002) due to its ability to fold secondary structures
covering Shine-Dalgarno sequence.
The several years of studies on structure and function of individual elements of mRNA
sequence resulted in a design of the optimum expression vector for the in vitro protein
expression systems. This has been exemplified by pIVEX (In Vitro EXpression) plasmid
(Betton 2003). This plasmid is characterized by its ability to form secondary structures at the
level of mRNA, particularly within the Shine-Dalgarno system and AUG initiation codon.
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Due to this, both fragments of mRNA sequence are exposed and they easily bind to a
ribosome. An integral part of the vector also includes the sequence of the bacteriophage
promoter, T7, which permits transcription of a given gene using T7 polymerase. Two types
of vectors are commercially available, including: (1) those containing His-tag sequences and
(2) Strep-tag, located on the amine or carboxylic terminus of the protein, which allows for an
easy and rapid purification of the biosynthesis products.

2.1 In vitro systems based on application of a semipermeable membrane
At present, the in vitro expression systems enjoy wide application due to the ease of
performing the reaction without the need to apply sophisticated equipment. However,
they exhibit low efficiency, not exceeding few tens of product nanograms in 50 µl reaction.
The solution which markedly increased protein expression level involved application of a
semipermeable membrane, used for the first time by Spirin (Baranov and Spirin 1993).
This produced a significant increase volume of the so-called feeding mix, containing free
amino acids, ribonucleotides and high energy molecules (mainly ATP and GTP), securing
in parallel high concentration of ribosomes with the yield produced in the reaction
compartment (Fig. 1, A and B). Application of a semipermeable membrane further
enabled a significant extension of the biosynthetic reaction since it could be continuously
supplied by inflow of indispensable reactants from the feeding mix (Fig. 1B). The
maximum duration of conducting the reaction averaged at approximately 30-50 h, with
plateau of reaction product being reached following around 30 h at 30°C. The system
produced a remarkable yield. For the first time milligram quantities of protein per 1 ml of
the reaction were obtained. The example involved expression of GFP (Green Fluorescence
Protein), the synthesis of which reached the level of around 5 mg in the course of a single
24 h (Fig. 2 A and B) reaction using the RTS (Rapid Translation System) (Iskakova et al.
2006). RTS is manufactured by Roche company and it has been designed in the basis of A.
Spirin’s patent (U.S. Pat. No. 5,478,730).
The RTS is based not only on the ingenious application of a semipermeable membrane but
also coupling the transcription and translation reactions, used also in the earlier designed
systems (Fig. 1A). Such an approach markedly abbreviated duration of the process and
reduced formation of nonspecific products since only the gene present in the expression
vector was undergoing transcription and, then, translation.
2.2 Advantages and drawbacks of RTS
The RTS system manifests several advantages. Due to release of ribosomes from the cell and
provision of appropriate conditions for the translation reaction, toxic proteins can be
produced. If using in vivo conditions, such toxicities factors would surely block living
processes in the cell. Enrichment of free amino acids with their radioactively labeled

substitutes permitted the effective labeling of nascent polypeptides.
The open nature of RTS systems and other in vitro techniques provided a handy tool for
studies on protein biosynthesis itself and on molecules such as antibiotics, which would
otherwise, of course block protein production. An interesting approach within RTS involves
screening analysis of known or potential antibiotics. Such an approach allows for a very
rapid determination of the inhibitory concentration of a given antibiotic (determination of
IC
50
) or preliminary analysis of the mechanism of antibiotic action.

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T7
polymerase
Ribosome
Semi-permeable membrane
upper fill port
lower fill port
Reaction
compartment
Feeding
compartment
magnetic stirrer
A
B
BIOSYNTHESIS PRODUCTTEMPLATES
Feeding compartment
(10 ml)
Reaction compartment

(1 ml)
supply
by-products
linear DNA
plasmid DNA
mRNA

Fig. 1. Production of proteins in RTS system. (A) Principles of the process; coupled
transcription-translation reaction runs on one of three templates in the reaction
compartment supplied by energy rich-components and amino acids from the feeding
compartment. (B) RTS reaction chamber with the semipermeable membrane separating
reaction and feeding compartments.
A typical marker protein used in studies on in vitro systems is GFP protein, due to the ease
of estimating the total product quantity and the fraction of active molecules (the
phenomenon of fluorescence). The GFP protein is almost ideal for this purpose since it
manifests a characteristic structure (Fig. 2A). The structure involves a barrel formed by 11 β
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structures and an active centre, the chromophore, located inside the molecule. The β
structures serve to protect the chromophore from the access of water molecules that could
otherwise block the fluorescence of the reaction (Yang, Moss, and Phillips 1996). The
appearance of inappropriate amino acids in GFP disturbs its structure and allows for
penetration of its interior by water molecules.
Using this system, it was possible to demonstrate that the in vitro system fully confirmed the
role of aminoglycosides as inducers of translation errors (Szaflarski et al. 2008). In the
presence of one of the aminoglycosides (streptomycin) expression of total GFP and active
fraction (i.e. GFP molecules inducing fluorescence) were followed by SDS polyacrylamide
gel electrophoresis (SDS PAGE) and native PAGE, respectively. The relations between

results read by these two techniques determined whether antibiotic (e.g. streptomycin)
impacted on the fidelity of translation reaction. If the active fraction of GFP decreases faster
than the total expression it means that full-size polypeptide chains are produced on the
ribosome however their protein folding failed due to increasing number of wrong amino
acids in the GFP and this protein was inactive (Fig. 2C). In view of the literature on
aminoglycoside character, this provided evidence for introduction of erroneous amino acids
to GFP molecule. This technique was demonstrated to be suitable to discriminate opposite
effects of edeine and pactamycin acting on the ribosome (Dinos et al. 2004).
Aminoglycosides were also tested in another in vitro synthesis system: the poly(U)-
dependent translation of polyphenylalanine (Szaflarski et al. 2008). In contrast to results
obtained in the RTS system, they failed to block the ribosome. However, following addition
of an additional leucine, it was found to be introduced to the polyphenylalanine chain
already at low concentration of streptomycin (around 1 µM). This reflected the similarity of
leucine codon and phenylalanine codon (UUC vs. UUU) and, thereafter, in situations
inducing elevated probability of translation errors, leucine was introduced instead of
phenylalanine. Thus, the observation did not allow a direct comparison between the two in
vitro translation systems. The RTS system resembles more closely the natural conditions
and, therefore, is more sensitive to action of antibiotics due to higher number of potential
targets.
Nevertheless, extracellular protein biosynthesis is linked to disadvantages which for several
years have been successively eliminated. In the course of studies the systems such as RTS
was found to support expression of relatively high amounts of protein but around half of
the proteins were found to be biologically inactive (Iskakova et al. 2006). However,
application of specific translation factors as EF-4 allowed reaching 100% efficiency of RTS
system (Qin et al. 2006).
The causes of lowered activity of proteins inside in vitro systems may be multiple but the
most probable one involves application of the bacteriophage polymerase T7, which is
exceedingly rapid. The transcription and translation are strictly interrelated during in vivo
conditions with elimination of the free space on mRNA between polymerase and the
ribosome. This prevents against development of spatial structures in mRNA molecule and it

does not allow for a precocious termination of translation. Application of T7 polymerase
disturbs the natural interrelationship between the polymerase and the ribosome, which may
lead to errors at the level of translation or to incorporation of inappropriate amino acids to
the growing polypeptide chain. The solution worked out by involved genetic recombination

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of the polymerase T7 of such a type that the enzyme contained two point mutations that
decreased the rate of activity (He et al. 1997). The results proved that despite the lowered
efficiency of the total biosynthesis the content of active proteins increased to almost 100%. A
similar effect was obtained decreasing temperature of the reaction. Most probably this
reflected the fact that, as compared to ribosomes, polymerase is much more temperature
sensitive, producing a slower rate of transcription and a similar pace of translation (Lewicki
et al. 1993).
3. Energy consumption and its regeneration inside in vitro systems
Protein biosynthesis represents a process of particular energetic requirements. In biological
systems the energy is obtained from hydrolysis of high energy bonds. For introduction of a
single amino acid to the growing polypeptide chain, the cell sacrifices as many as 10 high
energy bonds which is equivalent to hydrolysis of 10 molecules of ATP or GTP, each
characterized by bonding energy of ΔG
0
= -6 kcal/mol. The extreme energetic requirement
of a cell supporting protein biosynthesis explains development of sophisticated systems
which control energy loss. However, the systems retain the Achilles heel of contemporary
systems of protein biosynthesis in vitro in which their output remains seriously restricted by
excessive uncontrolled leaks of energy: usually not more than 5% of energy is expended to
support current protein biosynthesis while the remaining energy is wasted in uncontrolled
biochemical reactions. It should be borne in mind that injuring the cell we introduce an
extreme chaos to its metabolism. In contrast to in vitro conditions, in the in vivo conditions in

a bacterial cell as much as 70% of energy can be directed to support protein biosynthesis
(Szaflarski and Nierhaus 2007).
Therefore, in the techniques of protein biosynthesis in vitro a continuous replenishment of
high energy compounds (i.a. ATP and GTP), necessary for efficacious transcription and
translation reactions, continues to pose an enormous challenge. The earliest to be
designed system of replenishing the high energy compounds involved enrichment of the
cellular extract with millimolar concentrations of phosphoenolpyruvate (PEP), a
derivative of pyruvic acid, and with pyruvate kinase, which catalyzes transfer of a
phosphate group from phosphoenolpyruvate to AMP and ADP, yielding ADP and ATP,
respectively. Also GMP and GDP represent substrates for the kinase. PEP is distinguished
among all biologically active compounds by its content of the energetically most valuable
bond: the phosphoester bond contained in the compound carries the energy of ΔG
0
=-12
kcal/mol. Nevertheless, the PEP-based system carries also an extreme disadvantage: the
by-product formed during regeneration of the high energy molecules involves
orthophosphoric acid. The acid lowers pH of the reaction and, which is even more
important, it binds magnesium ions (Mg
2+
), markedly reducing their level in the reaction.
Mg
2+
stabilizes ribosome structure by its interaction with rRNA, therefore their reduced
level results in a disturbed ribosome structure and a reduced efficiency of protein
biosynthesis.
One of the ways in which the lowered concentration of Mg
2+
ions can be avoided involves
transformation of orthophosphoric to acetylphosphate using pyruvate oxidase and a
defined prosthetic group (TPP or FAD). The reaction requires an access of molecular

oxygen, the availability of which is restricted inside in vitro systems, particularly when the
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reaction is conducted in a few milliliter volumes. Nevertheless, the application of pyruvate
oxidase has significantly extend the duration of the effective protein biosynthesis reaction
which has markedly increased efficiency (Kim and Swartz 2000).
However, the above approach still hardly can be considered ideal. First of all, the restricted
access of molecular oxygen markedly reduces the potential for utilization of the system on a
larger scale. At present, the solution widely applied involves application of a combined
system, based on the traditional PEP/pyruvate kinase approach with acetyl phosphate
synthesis by acetyl-CoA, which allows for an effective elimination of free phosphoric acid
during synthesis of acetylphosphate from acetyl-CoA (Jewett and Swartz 2004). Application
of the system provided a breakthrough and permitted milligram quantities of the produced
protein in a volume of just one milliliter (Iskakova et al. 2006).

Fig. 2. Expression of GFP as a reporter protein in the presence of antibiotic streptomycin. (A)
The molecular structure of GFP with the internal chromophore as red (coordinates based on
PDB acc. no. 2B3Q). (B) The luminescence of GFP seen under UV lamp. (C) Parallel analysis
of GFP total expression (SDS PAGE) and its activity measured as its luminescence (native
PAGE). The graph demonstrates the fidelity (red line) of translation as the ratio between
total expression of GFP (blue dashed line) and the active fraction of GFP (green dotted line).
Increasing concentrations of streptomycin caused dramatic decrease in the ratio of the active
GFP to the total protein. Techniqual and experimental details see in Dinos et al. 2004;
Szaflarski et al. 2008; Qin et al. 2006.

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4. Biotechnological application of extracellular protein synthesis systems
4.1 Efficient expression of several proteins and their screening analysis in parallel
The extracellular protein expression provided the base for many automated techniques of
high-throughput expression and screening of several proteins in parallel (Angenendt et al.
2004; Spirin 2004). In such a system, using a single 96- or 384-well plate, multiple genes can
be copied, transcribed and translated in parallel, providing substrates for subsequent high-
throughput analysis of protein functions. The extensive scale of expression as well as the
rate of analysis warrant that the technique deserves to be considered in proteomics and
protein engineering based on screening analysis of gene and protein libraries as well as of
entire genomes and proteomes (He and Taussig 2007, 2008). The in vitro systems of protein
expression permit synthesis of protein population in a single reaction, which represents an
ideal and economic solution in complete screening analysis of proteins within a given gene
library (Chandra and Srivastava).
Such solutions can be proved by the technology known as in vitro expression cloning (IVEC)
(King et al. 1997). In the technique a large genomic library is preliminarily subcloned to
groups each containing 50-100 plasmids placed in the standard 96-well plate. They provide
a template for expression in an in vitro system. The plasmid-containing genes which yield
protein products are subsequently transferred by cloning to expression vectors, which allow
for synthesis of milligram quantities of proteins in RTS type in vitro systems.
The IVEC technique can further be improved by combining it with gene cloning and
amplification using the PCR reaction, thus eliminating the time-consuming cloning of the
genes to plasmids (Gocke and Yu 2009). Preparation of the appropriate primers containing
promoter sequences and Shine-Dalgarno sequences has facilitated protein synthesis directly
from products of the PCR reaction (Rungpragayphan, Nakano, and Yamane 2003). The
example includes an application of extracellular protein expression system for screening
analysis of the entire Arabidopsis thaliana genome in order to identify new genes and
products of their expression (Sawasaki et al. 2002).
Systems of in vitro translation have found application also in medical studies. The protein
truncated test (PTT) has been worked out in order to identify open reading frames (ORF)
(Roest et al. 1993). Detection of precocious translation termination within an ORF may reflect

mutation at the level of DNA, which provides grounds for distinguishing another genetically-
conditioned disease. Other applications are linked to production and analysis inside in vitro
systems of potential vaccines which, even if obtained on bacterial or animal ribosomes,
manifest the same immunological variables as those obtained in cultures of human cells
(Kanter et al. 2007). At present, new proteins representing potential anti-malaria vaccines have
been fully worked out in the systems of extracellular protein expression (Tsuboi et al. 2008).
Also the expression of virus-like particles (VLP) has been characterized inside in vitro systems
The examples include the phage protein, MS2 and C-terminal fragment of the protein core in
the hepatitis B virus (HBV), the biological activity of which has been identical to the forms
obtained in vivo (Bundy, Franciszkowicz, and Swartz 2008).
4.2 Production of proteins ”resistant” to expression
Considering the open character of in vitro systems due to the absence of biological
membranes, the systems could have been applied with excellent results for production of
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toxic or membraneous proteins, which manifest poor expression in in vivo systems (Jackson
et al. 2004). At present the in vitro systems allow for expression at a large scale of
membraneous proteins in modified extracts of E. coli (Klammt et al. 2006). Due to
application in the in vitro systems of techniques allowing for formation of disulphide
bridges, production of antibodies also has become possible. The example involves
immunoglobulin G (IgG), which when obtained in the system has proven to be fully
biologically active, manifesting affinity to antigen and stability identical to the natural
antibody (Frey et al. 2008).
4.3 Analysis of molecular interactions
Extracellular protein expression systems markedly facilitate the molecular analysis of
interactions between protein and X substance, where X may involve another protein, DNA,
RNA or a ligand (Jackson et al. 2004). In order to identify the interaction, one of the reactants
must be labeled (a protein, nucleotide or ligand) and placed in a system in which protein,

the other reactant is synthesized. Then, the arising complex is isolated from the reaction mix
using immunoprecipitation (Derbigny et al. 2000) or it may be directly analyzed in agarose
or polyacrylamide gels (Lee and Chang 1995).
4.4 Protein display technologies
The essence of protein display technologies involves establishing a link between genetic
information (genotype) and function of an unknown protein (phenotype) in the protein
library. The principal technique involves a ribosome display (He and Taussig 1997; Hanes
and Pluckthun 1997). Elimination of the STOP codon in mRNA permitted to obtain stable
complexes of mRNA-ribosome-protein. Thus, a kind of a frozen structure was obtained,
from the threshold of genetic world and proteomics. Subsequently, binding of the protein
formed on the ribosome to a defined ligand (which may involve also DNA or RNA) resulted
in development of an informational link between a given ligand and the sequence of protein
mRNA. Then, a given mRNA-ribosome-protein-ligand complex can be isolated by affinity
chromatography from the medium containing also other ligands while mRNA sequences
are identified by reverse transcription and DNA sequencing. In order to amplify efficacy of
the system, the process is conducted in a cyclic manner, i.e., the isolated mRNAs are
independently amplified and added again to the mixture of ribosomes and ligands, enabling
a more effective selection of an individual specific ligand. In combination with methods of
genetic engineering, including mutagenesis, the protein display technologies can be applied
not only in proteomics but also in molecular evolution studies. Now, the processes of
interactions between DNA, RNA and protein, which took millions of years of evolution may
be analyzed in the laboratory and their rate may be multiplied by selective amplification of
DNA.
5. Conclusion
Cell-free systems will be optimized and improved according to their expression yield,
protein specificity (“difficult proteins”) and protein folding. They will be more broadly
applied in protein microarrays technology where can be utilized for the analysis of protein-
protein interaction. Furthermore, protein technologies based on cell-free biosynthesis will be

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applied for protein engineering in order to synthesis specific antibodies or enzymes, as well
as for production of proteins for crystallisation.
The “post-genome” research requires comprehensive tool which will allow determination of
structure, function and specific location of the proteins in the network of proteomes. It has
to be performed effectively, quickly and on the multiple platform where large number of
proteins can be analyzed in the same time. Based on cell-free systems such analysis is
possible especially in the comparison to traditional cell-based systems where their
miniaturization is rather impossible.
6. Acknowledgements
This study was supported by the Polish Ministry of Science and Higher Education (grants
no. 0172/B/P01/2009/36). Authors thank to Dr. Jan Jaroszewski for his help in lingual
edition of the manuscript. Witold Szaflarski thanks to Prof. Knud H. Nierhaus for his long-
time powerful mentoring.
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